The present invention is directed generally to biosensors that are useful in the identification and analysis of biologically significant nucleic acids. The biosensors of the present invention and their applied methods provide a means for the direct analysis of nucleic acid hybridization and, therefore, have application to a myriad of biological fields including clinical diagnostics.
The detection and identification of microorganisms is a problem common to many areas of human and veterinary health. For example, the detection of pathogenic species such as Salmonella typhimurium, Listeria monocytogenes, and Escherichia coli, which are causative agents of major food borne epidemics, is a great concern within the food industry with respect to the quality and safety of the food supply. In other areas of human and veterinary health care, detection and identification of infectious diseases caused by pathogenic microorganisms and viruses is a first step in diagnosis and treatment. For example, it is estimated that 10-15 million office visits per year are for the detection and treatment of three major pathogensxe2x80x94Chlamydia ssp., Trichomonas vaginalis and Gradenerella vaginitis. Infections of these organisms annually effect 3.75 million, 0.75 million and 1.5 million patients, respectively.
Classical techniques routinely used for the detection and identification of microorganisms are often labor intensive, involving plating procedures which require lengthy analysis times. To illustrate, the method currently employed for the detection of Listeria monocytogenes in food and feed commodities involves a three stage analysis. The analysis begins with enrichment of the sample to be analyzed in a nutrient broth for 2 to 4 days. After the enrichment period, plating of the sample onto selective agar media is done and the sample is allowed to incubate for 2 days in order to obtain colonies for biotyping and serotyping, which may take as long as 20 days to complete (McLauchlin et al., 1988, Microbiology Review, 55: 578).
Detection processes based on culturing require analysis times which are too lengthy for effective monitoring and timely intervention to prevent the spread of biohazardous materials or treat disease. In addition, although these methods have been improved over the last decade, the chance of obtaining false negative results is still considerable, and many microorganisms are difficult to culture. Thus, plating/culture methods are limited with respect to their sensitivity, specificity, and lengthy analysis times that are required.
In order to shorten the time required to detect and identify pathogenic bacteria, viruses and genetic diseases, rapid tests such as enzyme immunoassays (EIA) have been developed (Olapedo et al., 1992). Although immunoassay techniques can be very sensitive and effective, there are practical drawbacks which have restricted the use of these methods. Such drawbacks include the need for highly skilled personnel, lengthy analysis and preparation times, and the large quantities of costly reagents that are required to do such analysis.
With the advent of nucleic acid amplification techniques (the polymerase chain reaction), the in-vitro amplification of specific sequences from a portion of DNA or RNA is now possible. Detection of very low numbers of microorganisms has been demonstrated (Rossen et al., 1991; Golsteyn et al., 1991; Wernars, K., et al., 1991). The polymerase chain reaction technique is sensitive and specific but involves complex manipulations in carrying out the tests and is not particularly well-suited for large numbers of samples. Due to the sensitivity of Polymerase Chain Reaction (PCR) technology, special rooms or areas for sample preparation and analysis are required to prevent contamination. In many tests PCR results must be confirmed by additional hybridization analysis. RNAs are difficult to assay by PCR but are very important for human viral detection. In general, PCR needs to be automated for acceptance as a practical diagnostic tool. Hybridization methods require as much as three or four days to complete results. Although the actual hybridization step can be as short as 18 hours, the entire detection process of a DNA/DNA hybrid can take as long as three days with a radioisotope marker.
Thus, there is a great need for simpler, faster and more cost-effective means for detecting specific biologically important RNA and DNA sequences in the fields of human and veterinary in-vitro diagnostics, food microbiology, and forensic applications.
Biosensors developed to date begin to overcome drawbacks associated with the current state of the art in detecting and identifying microorganisms. A biosensor is a device which consists of a biologically active material connected to a transducer that converts a selective biochemical reaction into a measurable analytical signal (Thompson et al., 1984. Trends in Analytical Chemistry, 3: 173; Guilbault, 1991, Current Opinion in Biotechnology, 2: 3). The advantages offered by biosensors over other forms of analysis include the ease of use (by non-expert personnel), low cost, ease of fabrication, small size, ruggedness, facile interfacing with computers, low detection limits, high sensitivity, high selectivity, rapid response, and reusability of the devices.
Biosensors have been used to selectively detect cells, viruses, other biologically significant materials, biochemical reactions and immunological reactions by using detection strategies that involve immobilization of enzymes, antibodies or other selective proteins onto solid substrates such as quartz and fused silica (for piezoelectric and optical sensors) or metal (for electrochemical sensors) (Andrade et al., 1990, Biosensor Technology: Fundamentals and Applications, R. P. Buck, W. E. Hatfield, M. Umana, E. F. Bowden, Eds., Marcel Dekker Inc., NY, pp. 219; Wise, 1990, Bioinstrumentation: Research, Developments and Applications, Butterworth Publishers, Stoneham, Mass.). However, such sensors are not widely available from commercial sources due to problems associated with the long-term stability of the selective recognition elements when immobilized onto solid surfaces (Kallury et al 1992, Analytical Chemistry, 64: 1062; Krull et al, 1991, Journal of Electron Microscopy Techniques, 18: 212).
An alternative approach is to create biosensors with long-term chemical stability. One such approach takes advantage of the stability of DNA. With the recent advent of DNA probe technology, a number of selective oligomers which interact with the DNA of important biological species, for instance salmonella, have been identified (Symons, 1989, Nucleic Acid Probes, CRC Press, Boca Raton, Fla.; Bock et al., 1992, Nature, 355: 564; Tay et al., 1992, Oral Microbiology and Immunology, : 344; Sherman et al., 1993, Bioorganic and Medicinal Chemistry Letters, 3: 469). These have been used to provide a new type of biorecognition element which is highly selective, stable, and can be easily synthesized in the laboratory (Letsinger et al., 1976, Journal of the American Chemical Society, 98: 3655; Beaucage et al., 1981, Tetrahedron Letters, 22: 1859; Alvarado-Urbina et al., 1981, Science, 214: 270).
Until recently, the only other research group in existence which has published work done on the fluorimetric detection of nucleic acid hybridization immobilized onto optical substrates is that of Squirrell et al. (C. R. Graham, D. Leslie, and D. J. Squirrell, Biosensors and Bioelectronics 7 (1992) 487-493.) In this work, single-stranded nucleic acid sequences ranging in length from 16-mer oligonucleotides to 204-base oligomers functionalized with an aminohexyl linker at the 5xe2x80x2 terminus were covalently attached to optical fiber sections functionalized with 3-aminopropyl triethoxysilane via a gluteraldehyde linkage. All investigations of nucleic acid hybridization were done by monitoring fluorescence intensity in an intrinsic mode configuration using complementary strands which had been previously labeled with a fluorescein moiety. This yielded a reusable assay system in which signal generation was observed to occur within minutes and nanomolar detection was achieved. However, this optical sensor technology developed by Squirrell et al. does not contain a transduction element which can transduce the binding event in a reagentless manner. For this assay to function, the target strands must be labeled prior to doing the assay in order for detection, making this technique unsuitable for practical applications.
Abel and co-workers (Abel, A. P.; Weller, M. G.; Duveneck, G. L.; Ehrat, M. and Widmer, H. M. Anal. Chem. 1996 68, 2905-2912) of Norvartis Ltd. (formerly Ciba-Geigy Ltd.) have recently reported an automated optical biosensor system. Their device utilizes 5xe2x80x2-biotinylated-16-mer oligonucleotide probes bound to an optical fiber functionalized with avidin to detect complementary oligonucleotides pre-labeled with fluorescein moieties in a total internal reflection fluorescence (TIRF) evanescent wave motif similar to that of Squirrell. Each assay consisted of a 3 minute pre-equilibration, 15 minute hybridization time, 10 minute washing procedure followed by a 5 minute regeneration cycle (chemical or thermal). A chemical denaturation scheme was observed to be the preferred embodiment for sensor regeneration as exposure of the oligonucleotide functionalized optical sensor to temperatures exceeding 52xc2x0 C. caused irreversible damage to the device, owing to denaturation of the avidin used for immobilization. This limitation renders the device function labile against sterilization techniques, such as autoclaving, and also indicates that rigorous cleaning of the sensor surface, such as by sonication, would also compromise the integrity of the sensor via denaturation of the affinity pair used to anchor the probe oligonucleotide. In order to detect nucleic acids not pre-labeled with fluorescein, and to overcome the limitation of Squirrell, a competitive binding assay was employed by Abel and co-workers. Detection of the unlabelled analyte was done by pre-treatment of the sensor with fluorescein labeled xe2x80x9ctracer-DNAxe2x80x9d followed by monitoring decreases in the fluorescence intensity of the sensor upon exposure to and subsequent displacement of the tracer-DNA by complement analyte nucleic acid. The dose-response curves reported by Abel et al. show a detection limit of 132 pmol (8xc3x971013 molecules) for this detection strategy. However, in addition to high detection limits and the inability of the device to withstand sterilization, this device cannot be classified as a biosensor technology due to the necessity for external treatment with tracer-DNA in order to achieve transduction.
The prior art with respect to patent literature contains many examples of xe2x80x9csensorxe2x80x9d devices which are based on nucleic acid molecules immobilized on waveguide supports and transduction strategies based on evanescent excitation. The technology of Gerdt and Herr (David. W. Gerdt, John. C. Herr xe2x80x9cFiber Optic Evanescent Wave Sensor for Immunoassayxe2x80x9d, U.S. Pat. No. 5,494,798) describes detection of nucleic acid hybridization based on alterations in the quantity of light transmitted from one optical fiber in a coupled fiber system (similar to that of a Mach-Zehnder interferometer) to the second fiber of the waveguide system. The quantity of light transferred is a function of the refractive index of the media on or surrounding the waveguides. Refractive index alterations affect the penetration depth of the evanescent wave emitted from the first waveguide into which optical radiation is launched. This standing wave of electromagnetic radiation subsequently propagates into (and thus transfers optical radiation to) the second waveguide. Therefore, the device is sensitive to refractive index alterations occurring within a volume surrounding the first waveguide with a thickness of ca. one wavelength of the light propagating within that waveguide. One of the arms of the waveguide may be functionalized with immobilized nucleic acid molecules which serves to provide selective binding moieties. The change in refractive index of the thin film of nucleic acids on the first waveguide upon the occurrence of hybridization with target nucleic acid sequences alters the quantity of light transferred to the second waveguide, thereby providing a means of signal transduction. Hybridization events may then be identified based on changes in the output ratios of the two waveguide arms in the coupled fiber system. One limitation of this technology lies in the fact that any alterations in refractive index near the surface of the waveguides will provide alterations in the output ratios of the two fibers. Therefore, non-specific binding events (such as protein adsorption) will provide false positive results.
In order to avoid the problem of interferents providing false positive results, a transduction strategy which is sensitive to the structure of the binding pair (i.e. recognition element and target) is required. The technologies of Fodor, Squirrell (David James Squirrell xe2x80x9cGene Probe Biosensor Methodxe2x80x9d International Application Number PCT/GB92/01698, International Publication Number WO 93/06241, International Publication Date: Apr, 1, 1993.), Sutherland et al. (Ranald Macdonald Sutherland, Peter Bromley and Bernanrd Gentile xe2x80x9cAnalytical Method for Detecting and Measuring Specifically Sequenced Nucleic Acid.xe2x80x9d European Patent Application Number 87810274.8, Publication Number 0 245 206 A1, Date of Filing: Apr, 30, 1987.), Hirschfeld (Tomas B. Hirschfeld, xe2x80x9cNucleic Acid Assay Methodxe2x80x9d U.S. Pat. No. 5,242,797, Date of Patent: Sept. 7, 1993.), and Abel et al. (Andreas P. Abel, Michael G. Weller, Gert L. Deveneck, Markus Ehrat, and H. Michael Widmer, Analytical Chemistry, 1996, 68, 2905-2912.) overcome this limitation by using fluorescent probes which associate with the binding pair or are attached to selective binding moieties capable of binding to a portion of the binding pair. These inventions provide methods to measure nucleic acid hybridization on waveguide surfaces based on evanescent excitation and TIRF. In each embodiment, an oligonucleotide probe capable of selective binding to a target sequence is covalently immobilized on a waveguide surface. For the cases of Squirrell and Abel et al., each define two preferred embodiments for the detection of hybridization events. The first embodiment of Squirrell and Abel et al. are essentially identical wherein the target nucleic acid is functionalized with a fluorescently detectable agent (by chemical or enzymatic methods) as a first step prior to detection. Upon hybridization between the labeled target and immobilized nucleic acid, the fluorescent agent is then bound in close proximity to the waveguide surface where it may be excited by evanescent wave formation and emission from the fluorophore collected and quantitatively measured. In the second preferred embodiment of Squirrell, hybridization between the immobilized oligonucleotide and the target sequence is first done. Subsequent to the first hybridization event, a fluorescently labeled oligonucleotide present in the system may then undergo hybridization with all or a portion of the remainder of the target sequence not hybridized with the immobilized sequence. The binding of the third (labeled) oligonucleotide provides a fluorescent species bound in close proximity to the waveguide which may furnish transduction via evanescent excitation and collection of the emitted radiation. In the second embodiment of Abel et al, a method for the detection of nucleic acids not pre-labeled with a fluorescent moiety via a competitive binding assay is described. Detection of the unlabelled analyte was done by first pre-treating the optical sensor with immobilized probe nucleic acid with fluorescein labeled xe2x80x9ctracer-DNAxe2x80x9d. The quantity of tracer-DNA may be monitored via the evanescent excitation and collection motif. Binding of the analyte could be followed by monitoring decreases in the fluorescence intensity from the sensor as a function of the displacement of the tracer-DNA via competitive binding with non-fluorescent analyte nucleic acid in a dose-response convention.
In the methods of Sutherland et al. and Hirschfeld, transduction of hybridization events is provided by fluorescent intercalating dyes (e.g. ethidium bromide). Following hybridization between the single-stranded target and immobilized probe nucleic acids, intercalant fluorescent dye molecules from solution insert into the base stacking regions of the immobilized double-stranded nucleic acid. An increase in the fluorescence quantum efficiency, fluorescence lifetime, stokes shift of the fluorescent intercalant probes often occurs upon association with double-stranded nucleic acid. It is claimed by the inventors that these enhanced features may be monitored by evanescent excitation and collection of fluorescence emission.
Fodor et al. have employed light-directed chemical synthesis to generate miniaturized, high density arrays of oligonucleotide probes. DNA oligonucleotide arrays have been fabricated using high-resolution photolithography in combination with solid-phase oligonucleotide synthesis. This form of DNA chip technology may be used for parallel DNA hybridization analysis, directly yielding sequence information from genomic DNA segments. Prior to sequence identification, the nucleic acid targets must be fluorescently labeled, either prior to or after hybridization to the oligonucleotide array, via direct chemical modification of the target strand or by use of an intercalant dye subsequent to hybridization on the DNA chip. The hybridization pattern, as determined by fluorescence microscopy, is then deconvolved by appropriate chemometric processing to reveal the sequence of the target nucleic acid. Rather than focusing on selective detection of trace quantities of a particular nucleic acid sequence, this technology has focused on sequence analysis of nucleic acids in suitably high copy number so as to sufficiently occupy the oligonucleotide array.
Notwithstanding the indubitable accomplishments of the aforementioned prior art, there yet exists limitations in these technologies for which further improvements are most desirous. Although the strategies employed by Sutherland et al. and Hirschfeld overcome the limitations of Gerdt and Herr with regard to signal origin and the generation of false positive results, these assay methods are limited by the amount of signal which can be generated by evanescent excitation. For multimode waveguides, less than 0.01% of the optical radiation carried within the waveguide is exposed to the outer medium in the form of an evanescent wave (R. B. Thompson and F. S. Ligler, xe2x80x9cChemistry and Technology of Evanescent Wave Biosensorsxe2x80x9d in Biosensors with Fiberoptics, Eds.: Wise and Wingard, Humana Press Inc., New Jersey,1991, pp.111-138.). In the case where monomodal waveguides are used, ca.10% of the radiation carried by the waveguide is exposed to the outer medium in the form of an evanescent wave (David. W. Gerdt, John. C. Herr xe2x80x9cFiber Optic Evanescent Wave Sensor for Immunoassayxe2x80x9d, U.S. Pat. No. 5,494,798). In the classic total internal reflection fluorescence (TIRF) evanescent wave configuration, the critical angle (xcex8c) for the waveguide/solution interface (xcex8cW/S) is larger than xcex8c for the waveguide/biological film interface (xcex8cW/B), only the evanescent component of the propagated radiation will enter the biological film. The principle of optical reciprocity states that light coupled back into a waveguide as a plane wave will be in the same way as the primary process when a plane wave generates an evanescent wave (Ranald Macdonald Sutherland, Peter Bromley and Bernanrd Gentile xe2x80x9cAnalytical Method for Detecting and Measuring Specifically Sequenced Nucleic Acidxe2x80x9d European Patent Application Number 87810274.8, Publication Number 0 245 206 A1, Date of Filing: Apr 30, 1987, p.13.). Thus, for the fluorophores excited by evanescent waves created from modes propagating at or near xcex8cW/S, none of the fluorescence emission can be coupled back into the waveguide in the same propagation mode as xcex8cW/S would be  greater than 90xc2x0 (U. J. Krull, R. S. Brown and E. T. Vandenberg, xe2x80x9cFiber Optic Chemoreceptionxe2x80x9d in Fiber Optic Chemical Sensors and Biosensors, vol.2, Ed. O. S. Wolfbeis, CRC Press, Boca Raton, 1991, pp.315-340.). Hence a large portion of the signal would be lost to the surroundings for systems in which fluorescence emission originates from thin films of a lower refractive index than that of the waveguide onto which they are immobilized. It has been shown by Love et al. that under optimal conditions, only 2% of the light emitted by the fluorophore in the medium of lower refractive index may be captured and guided by the fiber (W. F. Love, L. J. Button and R. E. Slovacek, xe2x80x9cOptical Characteristics of Fiberoptic Evanescent Wave Sensors: Theory and Experimentxe2x80x9d in Biosensors with Fiberoptics Eds.: Wise and Vingard, Humana Press Inc., New Jersey, 1991, pp. 139-180.).
The present invention concerns biosensors for direct detection of nucleic acids and nucleic acid analogs. The device comprises a light source, a detector, and an optical element for receiving light from the source and conveying it to an interaction surface of the optical element. A nucleic acid or nucleic acid analog for a particular nucleic acid sequence or structure (i.e. which is complementary to the target nucleic acid(s)), is immobilized onto the interaction surface of the optical element. Fluorescent ligands are provided that will bind into or onto the hybridized nucleic acid complex and fluoresce when stimulated by the light source. Subsequent to excitation by electromagnetic radiation of suitable wavelength bound within the optical element, the resultant fluorescence is collected within the optical element and guided to the detector to signal that the target nucleic acid(s) has complexed with the immobilized probe and thus indicate the presence of the target in the sample. An interaction surface is defined to mean a surface of the optical element on which nucleic acid is immobilized, and at which the fluorescent molecules interact with the light.
This invention provides biosensors in which the interaction surface is functionalized with nucleic acid probe sequences such that the index of refraction of the immobilized layer (Substrate Linker/Nucleic Acid/Fluorescent Ligand) is equal to or greater than the refractive index at the surface of the waveguide such that the organic coating becomes an extension of the waveguide. The index of refraction of the immobilized layer is dependent, at least in part, on the loading of immobilized molecules and linkers on the surface and the chemical nature of the immobilized molecules and any linkers.
Preferred biosensors which offer high-sensitivity and low-detection limits may be realized by activating the interaction surface of an optical element with substrate linker molecules of at least about 25 xc3x85 (Angstrom) in length followed by attachment of a selected probe nucleic acid sequence to that linker. (A probe nucleic acid is, at least in part, complementary to a target nucleic acid.) The preferred method for attachment of the probe nucleic acid to the substrate linker is by in-situ synthesis of the nucleic acid sequence onto the linker terminus using solid-phase nucleic acid synthesis methods or routine modifications of thereof. Such methods of in-situ synthesis are particularly useful for immobilization of nucleic acids of 50 or fewer bases and more particularly useful for nucleic acids of 30 or fewer bases.
The fluorophore may be tethered to the immobilized DNA, for example, by use of a hydrocarbon tether. The use of tethered probes can significantly reduce biosensor response time as the response mechanism is not diffusionally controlled. The associated fluorophore provides for internal calibration of optical source intensity and detector drift. It also provides for calibration of photobleaching, and provides for internal calibration by monitoring bound against free dye by use of, for example, time-resolved fluorescence measurements.
The optical element preferably comprises an optical waveguide which also conveys the fluorescent light to the detector. The optical waveguide preferably conveys the emitted light by total internal reflection to the detector. The optical waveguide can comprise an optical fiber, a channel waveguide, or a substrate that confines light by total internal reflection. The fluorescent molecules preferably provide sufficient Stokes shift such that the wavelength of the light source and the wavelength of the fluorescent light are easily separated. The fluorescent molecules can be provided in a solution in which the optical element is immersed, or by a tether to the nucleic acid that is immobilized to the linker.
In the practice of the present invention, the light source can be any suitable source such as a gas laser, solid state laser, semiconductor laser, a light emitting diode, or white light source. The detector can be any suitable detector such as a photomultiplier tube, an avalanche photodiode, an image intensifier, multi-channel plate, or semiconductor detector. The biosensor system can be a multi-wavelength, multi-fluorescent system. The light coupling of the system can also be modified to allow a multitude of disposable biosensors to be analyzed either sequentially or in parallel.
The biosensor system of the present invention can be constructed and used to detect each of a mixture of target nucleic acids (for example, Chlamydia and Gonorrhea in urogenital infections or E. coli and Salmonella during food processing). This may be done by using a plurality of fluorophores (which, for example, fluoresce at different wavelengths), each of which is tethered to an immobilized nucleic acid probe that is characteristic of or specific for detection of a given species or strain. In this example, the observed wavelength(s) of fluorescence emission will then be specific for hybridization of a given target nucleic acid to its complementary immobilized probe.
The biosensors of the present invention have an improved detection limit and sensitivity with respect to the prior art and are shown to be stable over prolonged storage and severe washing and sterilization conditions. Sensors stored over 1 year in vacuo, in 1:1 ethanol/water solutions, absolute ethanol, or dry at xe2x88x9220xc2x0 C. provide identical response characteristics to those freshly prepared. Adsorbed fluorescent contaminants accumulated through storage can be removed (as confirmed through fluorescence microscopy investigations) by sonicating the biosensors in 1:1 ethanol/water where the sensitivity of the device has consistently been observed to increase by a factor of c.a. 2.5 from this pre-treatment with respect to that of freshly prepared biosensors not cleaned before use. Unlike those of the prior art (e.g. Abel et al.), the optical biosensors of the present invention have also shown to be thermally stable wherein device function is maintained after sterilization by autoclaving (20 minutes, 120xc2x0 C., 4 atmospheres over-pressure). The ability to clean and sterilize a biosensor device so that it may be usable in an on-line configuration and/or in clinical applications is a significant advantage yet realized only by the technology reported herein. Biosensors of this invention also allow for more rapid sample analysis with improved response time for signal generation.
The present invention also provides a recyclable or disposable biosensor for detecting a target nucleic acid, which biosensor includes an optical element for receiving and conveying light to an interaction surface of the optical element and nucleic acid, for a particular nucleic acid sequence which is complementary to the target nucleic acid, immobilized onto the interaction surface of the optical element. The recyclable or disposable biosensor preferably comprises an optical waveguide, which preferably conveys the light by total internal reflection to the interaction surface of the optical waveguide when the organic coating is of equal or higher refractive index in comparison to the surface of the waveguide. The optical waveguide preferably comprises an optical fiber. Fluorescent molecules are provided in a solution in which the recyclable or disposable biosensor is immersed that will bind upon hybridization of the immobilized nucleic acid with complementary target nucleic acid and fluoresce when stimulated by light. Alternatively, the fluorescent molecules are provided bound by a tether to the immobilized nucleic acid.
The present invention provides biosensors for direct analysis of nucleic acid hybridization by use of an optical substrate such as an optical wafer or an optical fiber, and nucleic acids or nucleic acid analogs which have been immobilized onto the optical substrate. Generation of a fluorescence signal upon hybridization to complementary nucleic acids and nucleic acid analogs in a sample may be achieved in a number of different ways. Biosensors of this invention are sufficiently sensitive to directly detect very small quantities of target nucleic acids in a sample without the need to employ nucleic acid amplification methods such as PCR techniques. Biosensors of this invention can have detection limits for target nucleic acids below 108 molecules.
The optical biosensor comprises nucleic acid strands or nucleic acid analogs of a specific selected sequence immobilized onto activated optical supports. The selected immobilized sequences are capable of binding to target sequences, including sequences characteristic of and selective for viruses, bacteria, or other microorganisms as well as of genetic disorders or other conditions. Biosensors having such characteristic or selective immobilized sequences are useful for the rapid screening of genetic disorders, viruses, pathogenic bacteria and in biotechnology applications such as the monitoring of cell cultures and gene expression. One important avenue which has been widely ignored by the nucleic acid biosensor community is the investigation of multi-stranded (xe2x89xa73) nucleic acid formation. For example, triple-helical oligonucleotides have been reported to offer potential use as: sequence-specific artificial nucleases ({a} Moser, H. E.; Dervan, P. B. Science, 1987, 238, 645. {b} Strobel, S. A.; Doucettestamm, L. A.; Riba, L.; Housman, D. E.; Dervan, P. B. Science, 1991, 254, 1639.), DNA-binding protein modulators/gene expression regulators ({a} Cooney, M.; Czernuszewicz, Postel, E. H.; Flint, S. J.; Hogan, M. E. Science, 1988, 241, 456. {b} Durland, R. H.; Kessler, D. J., Gunnel, S., Duvic, M.; Pettit, B. M.; Hogan, M. E.; Biochem., 1991, 30, 9246. {c} Maher, L. J.; Dervan, P. B.; Wold, B.; Biochemistry, 1992, 31, 70. {d} Maher, L. J. BioEssays, 1992, 14, 807. {e} Maher, L. J. Biochemistry, 1992, 31, 7587. {f} Duvalvalentin, G.; Thoung, N. T.; Hxc3xa9lxc3xa8ne, C. Proc. Nat. Acad. Sci. USA, 1992, 89, 504. {g} Lu, G.; Ferl, R. J. Int. J. Biochem., 1993, 25, 1529.), materials for genomic mapping ({a} Ito, T., Smith, C. L.; Cantor, C. R. Proc. Natl. Acad. Sci. U.S.A., 1992, 89, 495. {b} Ito, T., Smith, C. L.; Cantor, C. R. Nucleic Acids Res. 1992, 20, 3524.), and highly selective screening reagents to detect mutations within duplex DNA (Wang, S. H., Friedman, A. E., Kool, E. T. (1995) Biochemistry 34, 9774-9784.). The present invention can also be used to detect the formation of multi-stranded nucleic acid hybrids (for example, formation triple-helical nucleic acids), and therefore could, for example, operate to monitor the effectiveness, dose dependence and intracellular concentration of nucleic acid pharmaceuticals used in gene therapy applications or as an assay to identify multi-strand formation associated with any of the aforementioned potential applications associated with triple-helical oligonucleotides.
The invention is a biosensor system for detecting a target nucleic acid, which consists of at least three layers, two of which are a waveguide, wherein one layer includes a nucleic acid or nucleic acid analog capable of hybridizing to the target nucleic acid, and wherein a fluorophore is tethered to the nucleic acid or nucleic acid analog and wherein the biosensor functions according to direct excitation. The invention also relates to a biosensor for detecting a target nucleic acid, which comprises an inner layer, a middle layer and an outer layer, wherein
the inner layer has refractive index n1,
the middle layer includes a nucleic acid or nucleic acid analog capable of hybridizing to the target nucleic acid and has refractive index n2, which is greater than or equal to refractive index n1, and
the outer layer has refractive index n3 which is less than refractive index n2.
and wherein a fluorophore is tethered to the nucleic acid or nucleic acid analog of the middle layer and wherein the biosensor functions according to direct excitation.
In a preferred embodiment, the inner layer is an optical fiber or optical wafer and the outer layer is an ambient. The outer layer is an aqueous based solution. The biosensor is useful for detection of triplex formation or multi-stranded nucleic acid formation. The triplex formation preferably involves a branched antisense nucleic acid which inhibits expression of a target nucleic acid sequence by triplex formation with the sequence.
The biosensor is useful for detection of nucleic acids of bacteria, viruses, fungi, unicellular or multicellular organisms or for the screening of nucleic acids of cells, cellular homogenates, tissues or organs.
Preferably, a fluorophore is tethered to a nucleic acid or nucleic acid analog which is one of the layers of a biosensor having at least three layers and the biosensor functions according to direct excitation. The invention also includes the use of a fluorophore for detecting a target nucleic acid.
The invention also relates to a method of detecting a target nucleic acid, comprising:
pre-treating a sample so that target nucleic acids characteristic of or selective for said sample are available for hybridization;
contacting the sample with the middle layer of the biosensor of claim 2, such that the target nucleic acids can hybridize to the nucleic acids or nucleic acid analogs of the middle layer;
allowing the fluorophore tethered to the nucleic acids of the middle layer to bind upon hybridization of the target nucleic acids with the nucleic acids or nucleic analogs of the second layer;
illuminating the fluorescent molecules with light such that fluorescence is stimulated; and
detecting the emitted fluorescence, whereby the presence of the target nucleic acid is detected.